In this project we will study the structure and function of biological members using fluorescence spectroscopy. We will focus on two aspects of the problem of membrane structure. In the first we will map out the spatial distribution of tryptophan residues in membrane proteins in order to obtain information about their structure. In the second we will investigate the nature of lateral organization in biological membranes by searching for heterogeneity in the distribution of lipids. In particular, we will: 1) Determine the tryptophan distribution in several membrane proteins including cytochrome b5 reconstitued into egg phosphatidylcholine vesicles, bacteriorhodopsin of Halobacterium halobium, band 3 in band 3 vesicles and band 3 reconstituted into egg phosphatidylcholine vesicles; 2) Further characterize the location and fluorescence behavior of the n-anthroyloxy fatty acid probes in membranes; 3) Expand our initial study of lipid domains to other membrane systems. Our studies of the structure of band 3 suggest that a considerable fraction of the protein mass lies outside the lipid bilayer. This also appears to be the case for bacteriorhodopsin and, if confirmed, would necessitate a considerable change in what is presently accepted as its structure. Our studies on lateral organization indicate that lipid domains are an important feature of membrane structure and since they exist to different degrees in different membranes probably play an important role in cellular function. Our studies using Tb fluorescence directly confirm that free fatty acids can alter protein structure, probably by altering a specific lipid domain.